Microbiological Experiment

LB Medium

Preparation of competent cells

1. Frozen strains at -70 ℃ are inoculated into culture dishes by streaking method, marked, and cultured overnight at 37 ℃.
2. The next day, a single colony is picked from the plate, inoculated into a tube containing 3 mL LB medium, and incubated overnight at 37 °C by shaking. The next day, 1 mL of the bacterial solution is inoculated into a 500 mL flask containing 100 mL LB medium, and incubated at 37 ℃ for about 2-3 h with severe shaking (200-300 rpm).
3. When the colony OD value of 600 nm reaches 0.3-0.4, remove the flask and place it on ice for 10-15 min. The bacterial solution is poured into a 50 mL centrifuge tube under aseptic conditions and centrifuged at 4,000 × g for 10 min at 4 ℃.
4. Discard the supernatant, invert the tube onto dry filter paper for 1 min, and blot the residual culture medium. Add 10 mL of 0.1 M CaCl2 to the centrifuge tube. Mix and suspend the thallus by shaking and allow to ice bath for 0.5 h.
5. Centrifuge at 4,000 × g for 10 min at 4 ℃, discard the supernatant, invert the tube onto dry filter paper for 1 min, and blot the residual culture medium. The bacteria are resuspended by adding 4 mL of 0.1 M CaCl2 precooled with ice. Each tube is divided into 0.2 mL, and stored at 4 ℃ for later use. The use effect is better within 24-48 h. If it is not used, it can be stored in the refrigerator at -70 ℃.

Transformation

1. Prepare the ice box in advance.
2. Take out DH5α bacteria from -80 ℃ refrigerator, take out the required plasmid from -20 ℃ refrigerator (the required concentration for transformation is generally 1 ng~10 ng/uL), and let it stand for 20 min until it is thawed.
3. Take the corresponding culture medium with antibiotics from the refrigerator and put it upside down in the incubator.
4. Operate in the ultra-clean table (wear laboratory clothes and gloves, keep leaning forward, and do not take out the equipment): use a pipette gun to blow the bacterial solution evenly, mix each plasmid solution and bacterial solution in a 1.5 mL EP tube at a ratio of 1:50, blow evenly, and write the label. All operations are carried out next to the alcohol lamp.
5. Insert the two tubes of mixed solution into ice and let it stand in the fume hood for 20 min.
6. Heat shock: the mixed solution is treated in a water bath at 42 ℃ for 1 min, then placed in a fume hood and placed on ice for 3 min.
7. According to the number of culture medium used, sterilize the coater with an alcohol lamp flame and set aside for use.
8. Take out the pre-treated culture medium and write down the label. Use a pipette gun to add the two tubes of mixed solution to the culture medium respectively (the outside side of the medium cap is up), then rotate the culture medium with the left hand, and apply the bacterial liquid evenly with the right hand. After covering the cap, the culture medium is inverted in the incubator for 16 h.

Monoclonal colonies selection and amplification

1.Take out the culture dishes prepared yesterday from the incubator, evaluate the situation of plasmid transfer and seal them with sealing film, and store them in the refrigerator at 4 ℃ for selection in the afternoon.
2. Prepare 8 shaker tubes with written labels, including 3 for selecting C1 single bacteria, 3 for selecting C2 single colonies, and 2 for selecting RTTA-P65 single colonies. Add 5 mL of Amp+ LB culture solution to the electric pipette for each shaker (the mouth and cap of the bottle containing the culture solution should be sterilized by alcohol lamp).
3. Select single colonies with a small pipette gun equipped with a gun tip (different areas should be selected when multiple colonies are selected on the same board). After selecting colonies, extend the gun tip below the liquid level in the corresponding shaking tube and blow it several times to mix evenly.
4. Seal the selected culture dishes with sealing tape and put them in the refrigerator at 4 ℃.
5. 8 shaker tubes are incubated at 37 ℃ and shaked at 220 rpm for 16 h.

The planet of the boy

The planet of the boy